Everything in 2x plus blanks. Team: the boys. Status: CDI-, CDI+ (2x), resistant (2x) have run overnight, results below. CDI- replicate + mixes to be done. Results: overnight growth of pure strains:
1b. Resistance rates
inhibitors (2x)
parallel cultures (5x)
fluorescent plate reader :-)
Fluctuation test on resistant strains, but making sure they are there for a reason (not just "happened to never meet a CDI+").
Everything in 2x would be good (we can then pool statistics). Team: the boys. Status: Done. Results: mutation rate for 49m is u ~ 10^-8, for 4945 u~10^-7.
2. CDI+ vs CDI-
titrate ratio CDI+ / CDI-: 1:1, 1:100, 1:10000
titrate cell density: 10^9 / ml, 10^7 / ml, 10^5 / ml (the latter might require some tuning)
when titrating cell density: take samples and measure cluster sizes under microscope
(later: develop model for cell density dependence)
(later: plus discuss toxin-independent cluster formation as an evolutionary strategy - might give a different angle on "selfish element" issue)
Everything in 3x would be good, or else 2x also ok. Team: the girls.
Protocol :
Status: tested under microscope, results being analyzed.
sector formation: CDI+ / CDI-, mix of two CDI+
Team: the boys. Status: unknown (please fill in).
competition assays with mixes of two CDI+
Team: M + JJ + S. Status: started cultures overnight.
single-cell competition in microbubbles
Team: F, JJ, Nick. Status: We got the bubbles and a single strain in it (RFP targets), and also both kinds (CDI+ green, CDI- red). The bacteria are taken from overnight culture, i.e. stationary phase:
We also got them together in a movie over an hour, but there is bleaching in the GFP channel:
The CDI+ bacteria are able to bind many CDI- and form tree-like structures:
We tried to grow the bacteria in the bubbles to observe non-stationary dynamics. It turned out to be more challenging than expected. We often get a lot of background fluorescence, so we used minimal medium + glucose instead of LB, There is still a large zoo of background noise levels, but the bacteria definitely grow a couple generations in the bubbles at 37 C:
BEFORE GROWTH
AFTER GROWTH
Results: droplets seem like an ideal environment to study single-cell competition dynamics of CDI without the complexity of 2D spatial structures of plates. There are, however, a few tricky points:
background noise might be hard to control (if the bacteria leak out GFP or other fluorescent metabolites, either dead or alive)
bleaching is an issue at single-cell level
bacteria swim in and out of focus
If we were to refine the experiments, we would:
get the right microscope filters (YFP, BFP)
use chromosomal fluorescent inserts, with strong primers
sort droplets at the end based on total fluorescence
The biological conclusions are:
CDI+ bacteria are much more sticky and tend to have larger cells than CDI-
one CDI+ bacterium can bind many targets, even during stationary phase
the efficiency of CDI-mediated killing at single-cell level might be low
Contact Dependent Inhibition (CDI) group page
Plan
1a. Growth
Everything in 2x plus blanks.
Team: the boys.
Status: CDI-, CDI+ (2x), resistant (2x) have run overnight, results below. CDI- replicate + mixes to be done.
Results: overnight growth of pure strains:
1b. Resistance rates
Fluctuation test on resistant strains, but making sure they are there for a reason (not just "happened to never meet a CDI+").
Everything in 2x would be good (we can then pool statistics).
Team: the boys.
Status: Done.
Results: mutation rate for 49m is u ~ 10^-8, for 4945 u~10^-7.
2. CDI+ vs CDI-
- titrate ratio CDI+ / CDI-: 1:1, 1:100, 1:10000
- titrate cell density: 10^9 / ml, 10^7 / ml, 10^5 / ml (the latter might require some tuning)
- when titrating cell density: take samples and measure cluster sizes under microscope
- (later: develop model for cell density dependence)
- (later: plus discuss toxin-independent cluster formation as an evolutionary strategy - might give a different angle on "selfish element" issue)
Everything in 3x would be good, or else 2x also ok.Team: the girls.
Protocol :
Results :
3. Single copy plasmid
- growth: 3 media (glu, gly, succ), 2 plasmid strains, CDI+(101), CDI-(105)
Team: JJ, MStatus: 7/30: prepared in above media, and growing.
- cell density: see above but with 1-copy plasmid
Team: A, S.Status: ongoing.
results:
4. The other cool things
- time-lapse microscopy (plasmid + patterning):
Status: tested under microscope, results being analyzed.- sector formation: CDI+ / CDI-, mix of two CDI+
Team: the boys.Status: unknown (please fill in).
- competition assays with mixes of two CDI+
Team: M + JJ + S.Status: started cultures overnight.
- single-cell competition in microbubbles
Team: F, JJ, Nick.Status: We got the bubbles and a single strain in it (RFP targets), and also both kinds (CDI+ green, CDI- red). The bacteria are taken from overnight culture, i.e. stationary phase:
We also got them together in a movie over an hour, but there is bleaching in the GFP channel:
The CDI+ bacteria are able to bind many CDI- and form tree-like structures:
We tried to grow the bacteria in the bubbles to observe non-stationary dynamics. It turned out to be more challenging than expected. We often get a lot of background fluorescence, so we used minimal medium + glucose instead of LB, There is still a large zoo of background noise levels, but the bacteria definitely grow a couple generations in the bubbles at 37 C:
- background noise might be hard to control (if the bacteria leak out GFP or other fluorescent metabolites, either dead or alive)
- bleaching is an issue at single-cell level
- bacteria swim in and out of focus
If we were to refine the experiments, we would:- get the right microscope filters (YFP, BFP)
- use chromosomal fluorescent inserts, with strong primers
- sort droplets at the end based on total fluorescence
The biological conclusions are: